Bead based hybridization assays have been reported for multiplexed detection of PCR amplified products. Each published method is well suited to a specific application. Published bead based hybridization assays require more than one step for hybridization, are time consuming and are not easily automated.
In a method disclosed by Brown et al, A Bead-Based Method for Multiplexed Identification and Quantitation of DNA Sequencs Using Flow Cytometry, Applied and Environmental Microbiology, October: 4258–4265, (2000), a double stranded amplified DNA is enzymatically digested with Shrimp alkaline phosphatase (SAP) and Exonuclease1. The single stranded DNA produced from the digestion is then annealed to an oligonucleotide on a bead and the hybridized product is detected with the Luminex flow analyzer.
Weiner et al, Multiplexed Single Nucleotide Polymorphism Genotyping by Oligonucleotide Ligation and Flow Cytometry, Cytometry 39:131–140 (2000), describes a universal ZipCode hybridization method. In this method, a complementary ZipCode sequence is attached to a long piece of DNA that is attached to a spacer used to reduce the stearic hindrance. A part of the allele complementary sequence is attached to the ZipCode sequence. A capture probe is then prepared by hybridization and ligation of the allele complementary sequence and a fluorescently labeled allele complementary sequence in the presence of the single stranded amplified DNA and ligase enzyme.
In Suspension Arrays for High Throughput, Multiplexed single Nucleotide Polymorphism Genotyping, Cytometry 40:102–108 (2000), Muzumder et al describes a more simplistic, but difficult approach. Their approach denatures a fluorescently labeled denatured PCR product and directly hybridizes that product to complementary oligonucleotides attached to beads.
Each of the above mentioned assays take on average several hours to go from amplification to detection. Holland et al in, Detection of specific polymerase chain reaction product by utilizing the 5′ - - - 3′ exonuclease activity of Thermus aquaticus DNA polymerase, Proc Natl Acad Sci USA 88 (16):7276–80, (1991), discloses real time PCR amplification. This method is generally referred to as Taqman® real time detection and is routinely used for real-time PCR amplification. Real time PCR amplification is measured using fluorogenic probes that can detect the 5′-exonuclease activity of the DNA polymerase. At present, Taqman® real time detection can simultaneously detect a maximum of four nucleic acid targets.